Cell cycle arrest and apoptosis of leukemia cells induced by L-asparaginase.

Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L-asparaginase is an event that is associated with the cell cycle arrest in G1 phase.

Chemical modification of L-asparaginase with comb-shaped copolymer of poly(ethylene glycol) derivative and maleic anhydride.

L-asparaginase from Escherichia coli, an antitumor enzyme, was chemically modified with a comb-shaped copolymer of poly(ethylene glycol) derivative and maleic anhydride (activated PM). The PM-modified asparaginase lost the immunoreactivity with retaining high enzymic activity and also prolonged the clearance time in blood. Intraperitoneal administration of PM-asparaginase markedly increased the mean survival-time of lymphoma L5178Y-bearing mice in comparison with that of unmodified asparaginase. Pretreatment of mice with PM-asparaginase before immunizing with unmodified asparaginase extremely suppressed the anti-asparaginase antibody production.

Biomedical and biotechnological applications of PEG- and PM-modified proteins.

Chemical modification of proteins and other bioactive molecules with polyethylene glycol (PEG) or its derivatives (PM) can be used to tailor molecular properties to particular applications, eliminating disadvantageous properties or conferring new molecular functions. Complexes of therapeutic proteins and PEG or PM show reduced immunoreactivity, prolonged clearance times and improved biostability. Modification with PEG can also increase the solubility and activity of enzymes in organic solvents, thus extending their potential for application in organic syntheses and biotransformation processes.

Preparation and application of a pentamannosyl monophosphate-bovine serum albumin conjugate.

Pentamannosyl monophosphate, derived from Hansenula holstii O-phosphomannan, was conjugated to bovine serum albumin by reductive amination. The conjugate inhibited the binding of the porcine testis mannose 6-phosphate receptor to the insoluble phosphomannan core. A mannose 6-phosphate receptor with a molecular weight of 200,000 was purified from porcine liver membranes, using an affinity matrix of the conjugate attached to Sepharose 4B. Rabbits were immunised with the conjugate, and the antisera were purified on a phosphomannan core-Sepharose 4B column in order to give an antibody which was specific for the 6-phosphate group and the equatorial HO-4 of D-mannose 6-phosphate. On Western blot analysis using the purified antibodies, ovalbumin, which contained a typical high-mannose type of oligosaccharide, was not recognised. However, a testicular glycoprotein fraction formed an immunostaining band. These results indicate the effectiveness of the conjugate as a ligand for mannose 6-phosphate receptors. The antibodies highly specific for mannose 6-phosphate may be used to detect or purify lysosomal enzymes.

Inhibition of Df-protease associated with allergic diseases by polyphenol.

It was reported that Df-protease from house dust mite (Dermatophagoides farinae) catalyzes the activation of the kallikrein-kinin system in human plasma and is closely associated with mite-induced allergy. Therefore, to prevent the release of kinin by Df-protease, the inhibitory activity of polyphenols including catechins and flavonols was tested in vitro and in vivo. Among them, myricetin and epigallocatechin gallate (EGCg) effectively inhibited the amidase activity of Df-protease with K(i) values of 1 x 10(-)(8) and 6 x 10(-)(4) M, respectively. The kinin release in human plasma was extensively inhibited by the addition of EGCg in comparison with myricetin. Enhancement of vascular permeability in guinea pigs caused by Df-protease was markedly suppressed by EGCg.

Stabilization of L-asparaginase modified with comb-shaped poly(ethylene glycol) derivatives, in vivo and in vitro.

L-Asparaginase from Escherichia coli was coupled with two types of comb-shaped copolymer of poly-(ethylene glycol) derivative and maleic anhydride (activated PM), having molecular weights of 13,000 and 100,000 (activated PM13 and PM100, respectively) with multivalent reaction sites. After single intravenous injections of PM100-asparaginase and nonmodified asparaginase into rats, the enzymic activity of PM100-asparaginase in serum was well retained for at least 11 days, and the serum L-asparagine concentration remained undetectable for 27 days. The half-lives of PM100-asparaginase and nonmodified asparaginase were 50 and 1.5 h, respectively. Stabilization of L-asparaginase toward heat, urea, and acidity was caused by modifying the enzyme with activated PM13 and PM100. Especially, PM100-asparaginase retained high enzymic activity toward heat and urea, compared with PM13-asparaginase. It was suggested that these modifiers with a comb-shaped form and with multivalent reactive sites cover the whole surface of the asparaginase molecule and stabilize its conformation possibly through multiple covalent bindings and through various noncovalent interactions.

Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol.

Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution.

Reduction of immunoreactivity of bovine serum albumin conjugated with comb-shaped polyethylene glycol derivatives.

Bovine serum albumin (BSA) was chemically modified with two types of comb-shaped copolymers of polyethylene glycol derivative and maleic anhydride, one with the molecular weight of 13,000 (activated PM13) and the other with 100,000 (activated PM100), to form PM13- and PM100-BSA. The immunoreactivity of BSA was markedly reduced by coupling with each modifier and was completely lost when 30% or 20% of amino groups in BSA were modified with activated PM13 or PM100, respectively. The esterase activity of PM13- and PM100-BSA without immunoreactivity were retained 63% and 93% of non-modified one, respectively. These results were discussed with those of modified-asparaginases(1).

Tolerogenic capacity of poly(ethylene glycol) (PEG)--modified ovalbumins in relation to their immunoreactivity towards anti-ovalbumin antibody.

Ovalbumin (OVA) was chemically modified with poly(ethylene glycol) (PEG) derivatives: activated PEG2, 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine and activated PM13, copolymer of poly(oxyethylene) allyl methyl diether and maleic anhydride. Pretreatment of BALB/c mice with PEG2- or PM13-OVA suppressed the production of both IgG- and IgE-class anti-OVA antibodies induced by subsequent immunizations with unmodified OVA. PEG2-OVA had higher potency to suppress OVA-specific immune response than PM13-OVA. Although extensive modification (62%) of amino groups in the OVA molecule with activated PEG2 was required to diminish most of its immunoreactivity towards anti-OVA antibodies, only a low degree of modification (27%) was sufficient to induce tolerogenicity to OVA. Thus, the loss of immunoreactivity of PEG2-OVA was not prerequisite for its tolerogenic capacity. Moreover, the use of completely denatured PEG2-OVA immunogen lead to the loss of its ability to induce immune tolerance to native OVA. These observations are discussed in relation to the regulatory mechanism of immune response.

Polyethylene glycol-modified concanavalin A as an effective agent to stimulate anti-tumor cytotoxicity.

The jack bean lectin, concanavalin A (Con A), was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine, activated PEG2, to form PEG-Con A. The immunoreactivity of PEG-Con A towards anti-Con A antibodies was reduced by increasing the degree of modification of amino groups in the Con A molecule. PEG-Con A had a complete reduction of the immunogenicity in mice and prolonged the clearance-time in blood. Although the mitogenic activity of Con A towards murine spleen cells was reduced by the conjugation with activated PEG2, the administration of PEG-Con A to mice enhanced the anti-tumor cytotoxicity of peripheral lymphocytes against melanoma B16 cells.

Alcoholysis of epsilon-decalactone with polyethylene glycol-modified lipase in 1,1,1-trichloroethane.

Lipase from Pseudomonas cepacia was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine, activated PEG2, to form PEG-lipase. The PEG-lipase is soluble and active in organic solvents. It catalyzes alcoholysis of racemic epsilon-decalactone with ethanol in 1,1,1-trichloroethane to form (R)-hydroxydecanoic acid ethyl ester. No alcoholysis of (S)-decalactone takes place. These results were discussed in relation to carbon number of n-alcohol, optimum temperature and comparison with modified and non-modified lipases.

Polyethylene glycol-modified pokeweed mitogen (PWM) as a potential non-immunogenic stimulator of lymphokine-activated killer cells.

Among the various plant lectins, pokeweed mitoge (PWM) is most effective in enhancing the cytotoxicity of human lymphokine-activated killer (LAK) cells. However, the use of PWM in adoptive immunotherapy has been limited due to the strong immune response against the protein of plant origin. Amino groups in PWM was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chioro-s-triazine, activated PEG2, to form PEG-PWM conjugates. Its immunoreactivity towards anti-PWM antibodies was reduced by increasing the degree of modification of amino groups in PWM. PEG-PWM, in which 54% of amino groups in PWM was modified with activated PEG2, had a nearly complete reduction of immunoreactivity. Intraperitoneal administration of PEG-PWM to mice did not produce substantial levels of anti-PWM antibodies. Nevertheless, PEG-PWM retained the ability to induce the maximum levels of cytotoxicity of human LAK cells in vitro.

Hemin (Fe(3+))-- and heme (Fe(2+))--smectite conjugates as a model of hemoprotein based on spectrophotometry.

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.

Suppression of ischemic edema in mice by manganese-hyaluronate conjugate.

Manganese-hyaluronate conjugate (Mn-HA) was synthesized from a diethylenetriaminepenta-acetic acid derivative of hyaluronic acid and manganese ion. The conjugate markedly scavenged super-oxide anion in vitro and exhibited much higher anti-inflammatory activity than superoxide dismutase in suppressing paw edema in mice when intravenously injected 30 min before the initiation of ischemia.

Immune tolerance induced by polyethylene glycol-conjugate of protein antigen: clonal deletion of antigen-specific Th-cells in the thymus.

Polyethylene glycol (PEG) conjugates of protein antigens induce antigen-specific immune tolerance of helper T (Th)-cells. However, the mechanism of this Th-cell tolerance has remained unelucidated. Using transgenic mice with ovalbumin (OVA)-specific T-cell receptor (TCR) genes, we examined the response of OVA-specific Th-cells towards tolerogenic PEG-conjugate of OVA in vitro and in vivo. When stimulated with PEG--OVA in vitro, transgenic OVA-specific Th-cells proliferated and produced interleukin 2, the levels of which were comparable to those induced by unmodified OVA. In contrast, PEG--OVA administered into the circulation of transgenic mice induced unresponsiveness in peripheral OVA-specific Th-cells. Moreover, in the thymus of these transgenic mice, the frequency of immature CD4+CD8+ (double positive) thymocytes was reduced. A similar phenomenon was not observed in transgenic mice treated with unmodified OVA. As autoreactive T-cells are known to be clonally deleted at the immature double positive stage in the thymus. Th-cell tolerance induced by PEG--protein antigens is at least in part mediated by central tolerance in the thymus, and is likely caused by the markedly enhanced stability of PEG--protein conjugates in the circulatory system.

Hydrogen gas evolution and carbon dioxide fixation with visible light by chlorophyllin coupled with polyethylene glycol.

Chlorophyllin a was conjugated with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene), PEG-NH(2), to form the PEG-chlorophyllin conjugate through acid-amide bonds. The PEG-chlorophyllin conjugate was stable toward light illumination under anaerobic condition in comparison with chlorophyllin a. The conjugate catalyzed the reduction of methyl viologen in the presence of 2-mercaptoethanol and the evolution of hydrogen gas in the presence of methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor) and hydrogenase (Scheme 1). Furthermore, the PEG-chlorophyllin conjugate catalyzed the photoreduction of NADP(+) or NAD(+) in the presence of ascorbate as an electron donor and ferredoxin-NADP(+) reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by the PEG-chlorophyllin conjugate under the illumination, CO(2) fixation was accomplished by the synthesis of malate (C(4)) from pyruvate (C(3)) and CO(2) in the presence of malic enzyme (Scheme 2). These reactions mentioned above did never proceed in dark or without each enzyme.

Photostable chlorophyll a conjugated with poly(vinylpyrrolidone)-smectite catalyzes photoreduction and hydrogen gas evolution by visible light.

Chlorophyll a was adsorbed to a synthetic smectite intercalated by poly(vinylpyrrolidone) (PVP) to form the chlorophyll-PVP-smectite conjugate (Chl-PVP-SME) having an absorption maximum at 677 nm. The conjugate was found to be stable toward light illumination in comparison with chlorophyll-smectite, chlorophyll-PVP, and free chlorophyll a. Chl-PVP-SME had a photoinduced activity for catalyzing the reduction of methyl viologen. Furthermore, the evolution of hydrogen gas was observed when an aqueous suspension containing Chl-PVP-SME, methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor), and hydrogenase was illuminated by visible light.